Members of the genus Rickettsia are the etiologic agents of rocky mountain and other spotted fevers and endemic, scrub, and epidemic typhus, diseases that pose a genuine health threat worldwide. Rickettsia prowazekii, the causative agent of epidemic typhus, like all members of this genus, is an obligate intracellular parasitic bacterium that can grow only within the cytoplasm of a eucaryotic host cell. The ability of the rickettsiae to successfully exploit this intracellular niche in animals as diverse as arthropods and humans and to subsequently cause serious human disease, provides the impetus for the study of these novel bacteria. The continuing goal of this research is to elucidate the genetic and biochemical processes that are involved in the successful intracellular parasitism of eucaryotic cells by R. prowazekii. In Specific Aim 1 our goal is to capitalize on a rickettsial transformation system and to identify and use genetic techniques to examine and manipulate the rickettsial genome. Experiments will be performed to optimize the conditions for transformation of the rickettsiae by electroporation and the identification or construction of suitable vectors. Transformation will be used to introduce altered rickettsial genes into R. prowazekii to generate specific rickettsial mutants by insertional inactivation techniques. In a similar manner, the feasibility of creating reporter gene fusions between rickettsial chromosomal genes and the lux genes coding for luciferase will be examined. Transformation will also be used to isolate and characterize virulence-associated genes. In Specific Aim 2 we shall examine expression of selected R. prowazekii gene linkage groups using RNase protection to identify and quantitate rickettsial transcripts. Organization of these genes into operons will be investigated and rickettsial transcriptional terminators will be characterized. The linkage groups to be examined include the rickettsial homolog of the major macromolecular synthesis operon, the genes contiguous to sdhA, and the genes coding for subunits of core RNA polymerase. These linkage groups code for products involved in replication, transcription, and translation as well as energy and biosynthetic metabolism.